Factor X activation by the extrinsic pathway is considered pivotal for the initiation of blood coagulation in normal hemostasis. The enzyme complex (extrinsic Xase) that catalyses this reaction is composed of the serine protease, factor VIIa, and the integral membrane protein, tissue factor, that associate tightly on a cellular or phospholipid surface in the presence of calcium ions. The exposure of cells bearing TF to flowing blood leads to the recruitment of factor VII, its proteolytic conversion to VIIa, the activation of factors X and IX and the subsequent down- regulation of the enzyme complex by inhibitors. The aims of this proposal are designed to investigate critical aspects of the initiation and regulation of the extrinsic pathway that remain poorly understood. Steady state and rapid kinetic measurements will be used to examine the regulation of factor Xa formation by Tissue Factor Pathway Inhibitor (TFPI). We will provide a complete kinetic and thermodynamic description of the regulation of active factor Xa produced by this pathway as a result of the ability of TFPI to function as a potent product-dependent feedback inhibitor of the catalyst. By comparing the functions of TFPI with antithrombin III, we will assess the dominant inhibitory pathway in extrinsic pathway control as well as the regulatory advantage conferred by the unique mechanism of TFPI action. We will dissect the molecular mechanisms of the activation of factor VII by the use of rapid chemical quench and a highly specific inhibitor of factor Xa. Finally, the kinetics of bond cleavages in factor IX leading to Ixabeta formation will be investigated to provide a quantitative explanation for the ability of factor IX to modulate the activation of factor X by the extrinsic pathway. These approaches will provide a kinetic and thermodynamic foundation for an understanding of the regulation of extrinsic Xase function with implications for normal hemostasis as well as thrombosis in vascular disease.